While protein-tyrosine kinases share a region of sequence identity corresponding to their kinase domains, the specific elements essential for catalysis, substrate binding and substrate specificity are largely undefined. The P130gag-fps transforming protein of Fujinami avian sarcoma virus is a cytoplasmic tyrosine kinase with a complex structure that includes a C-terminal kinase domain. To identify the precise N-terminal border of the v-fps catalytic region and to assess its interactions with non-catalytic domains, C-terminal v-fps polypeptide fragments of decreasing size were expressed in E. coli as trpE-v-fps hybrid proteins. All such polypeptides containing 263 or more residues derived from the C-terminus of P130gag-fps (i.e. residues 920-1182) were enzymatically active as tyrosine kinases. They autophosphorylated at physiological sites in vivo and phosphorylated exogenous substrates such as enolase and poly(glu,tyr) at tyrosine in vitro. Deletion of a further five amino acids from P130gag-fps residues 920-925 abolished all enzymatic activity. This deletion coincides with the predicted N-terminus of the v-fps ATP-binding site at residue 922. These data indicate that the N-terminal border of the ATP-binding site defines the start of the minimal v-fps tyrosine kinase catalytic domain, and show that this minimal domain is competent to bind substrates. More N-terminal non-catalytic sequences appear to functionally interact with the catalytic domain.